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【摘要】 目的 探讨肾脏缺血后处理对兔急性肠道缺血再灌注损伤的保护作用及其可能的机制。方法 健康新西兰大白兔30只,随机分为3组(每组10只):对照组(Con)、肠道缺血后处理(IIP)、肾脏缺血后处理(RIP)。再灌注结束后测定血浆中丙二醛(MDA)含量;实验结束后光镜下观察肠道组织形态学变化以及测定肠道组织髓过氧化物酶(MPO)活性。结果 IIP和RIP组再灌注2 h后MDA的含量明显的低于Con组(P<0.01); SIP和RIP组再灌注2 h后组织MPO的活性明显的低于Con组(P<0.01); SIP和RIP组再灌注2 h后光镜下观察肠道组织形态学变化证实肠道损伤程度的明显的轻于Con组(P<0.01)。结论 肠道缺血再灌注前肾脏短暂缺血再灌注对肠道缺血再灌注损伤具有显著保护作用。这种远离器官缺血后处理对肠道保护作用可能与减轻活性氧的损伤及抗氧化作用加强有关。
【关键词】 远隔缺血后处理;缺血再灌注;急性肠缺血
Impact of remote postconditioning on rabbits with acute intestinal ischemia-reperfusion injury
CHUYong-xin, YANGMu, ZHANGju-wen.
Department of Vascular surgery,Yantai Yuhuangding Hospital,the Affiliated Hospital of medical College,Qingdao University,Shandong 264000,China
【Abstract】 Objective In this study, we tested the hypothesis that remote postconditioning induced by a single 5-min episode of renal artery occlusion and reperfusion applied immediately before the onset ofsuperior mesenteric artery reperfusion protects the intestinal from reperfusion injury. Methods 30 healthy New Zealand white rabbits were randomly divided into three groups(n=10 in each group): control, intestinal ischemic postconditioning (IIP), and renal ischemic postconditioning (RIP). Tissue myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were determined and the morphology of some intestinal tissues was observed by microscopy at the end of the experiment. Results MDA levels and MPO activity was significantly reduced in IIP and RIP as compared to control (P<0.01),also was confirmed by the score of intestinal mucosal injury. Conclusion Renal postconditioning provides potent intestinal mucosal injury. The potential mechanism of remote postconditioning might be associated decreasing the injury caused by oxygen free radicals and strengthening the action of antioxidation.
【Key words】 Remote postconditioning;Ischemia - reperfusion injury;Acute intestinal ischemia
DOI:10.3760/cma.j.issn 1673-8799.2010.11.46
作者单位:264000青岛大学医学院附属烟台毓璜顶医院血管外科
越来越多的肠系膜上动脉栓塞和血栓形成早期发现早期手术治疗肠缺血再灌注损伤越来越受到临床医生的重视。缺血预处理和缺血后适应均可减轻缺血再灌注的损伤。但无法预知组织及器官缺血具体时间,缺血预处理的临床应用受到了极大限制。缺血后适应的实施直接作用于已发生缺血的重要器官,在临床上不适用于对缺血极其敏感的器官。2005年Kerendi[1]等利用小鼠心肌缺血模型,在心脏缺血再灌注前对肾动脉进行单次5 min的闭塞和1 min再通,发现可显著减轻心肌缺血再灌注损伤,从而提出了远隔缺血后处理即:组织器官缺血再灌注前在非本组织器官短暂的缺血,能减轻缺血再灌注的损伤。本实验研究肠道缺血再灌注前肾动脉短暂缺血再灌注即肾脏缺血后处理对兔肠道缺血再灌注的影响,为临床寻找减轻肠道缺血再灌注损伤更佳方法提供实验依据。
1 材料和方法
1.1 实验动物
健康新西兰大白兔30只,雌雄不拘,体重1.5~2.0 kg。所有动物随机分为3组(每组10只):对照组(Con)、肠道缺血后处理组(IIP)、肾脏缺血后处理组(RIP)。
1.2 方法
1.2.1 手术过程 所有新西兰兔术前12 h禁食,自由进水。称重后左下腹腔内注射10%水合氯醛(3 ml/kg)麻醉。上腹正中切口,长约10 cm,进腹后予肠系膜根部仔细分离显露肠系膜上动脉,再仔细分离显露左肾动脉,用无创小血管夹阻断动脉,分组干预:对照组:阻断肠系膜上动脉30 min,再灌注120 min; 肠道缺血后处理组:阻断肠系膜上动脉30 min 后,灌注1 min,阻断1 min,反复3次,继而在灌注到120 min; 肾脏缺血后处理组: 阻断肠系膜上动脉24 min 时,阻断左肾动脉5 min,开放肾动脉1 min,再灌注肠系膜上动脉120 min。
1.2.2 标本采集 再灌注120 min后直视下下腔静脉穿刺取血5 ml,血标本收集后,常温下静置30 min, 3000r/min离心15 min,分离的血浆保存于-20℃冰箱,待所有标本收集完后一次性检测。分光度计测定血浆MDA水平,血浆MDA含量以每毫升血浆所含的数量来表示(mmol/g)。在无菌原则下取距回盲部约20 cm的近端小肠组织5 cm左右,切除的近端2.5 cm肠组织冷冻贮存于-80℃,分光度计测定小肠组织MPO活性,并以每百克组织所含的单位来表示(U/100 g)。试剂盒均购于南京建成生物工程研究所。远端2.5 cm肠组织用40 g/L甲醛溶液固定,用石蜡包埋,切片,苏木精-伊红染色行组织病理学检查。肠组织病理评分采用双盲法按Chiu6级[2]评分法分析。
1.2.3 统计学方法 采用SPSS 11.0软件进行统计学处理,计量资料采用t检验,组间比较采用单因素方差分析,P<0.05为差异有统计学意义。
2 结果
2.1 将各组血浆中MDA的含量和组织中MPO的活性测定严格按照试剂盒说明进行操作,双盲法测得的肠壁损伤病理评分,见表1。SIP组和RIP组的MDA含量、MPO的活性及肠黏膜损伤评分与Con组比较,差异均有显著性(P<0.01)。SIP组与RIP组的MDA含量、MPO的活性及肠黏膜损伤评分比较无显著差异(P>0.0 5)。
3 讨论
本实验发现兔小肠急性缺血再灌注前给予肾动脉短暂缺血再灌可明显减轻小肠的缺血再灌注损伤。Li[3]等在远隔缺血后处理对心肌缺血的保护实验中发现丙二醛活性显著降低,提示远隔缺血后处理可减轻缺血心肌的过氧化反应。本实验血浆MDA含量较对照组明显减少,提示肾脏缺血后处理可减轻小肠缺血再灌注损伤的脂质过氧化,间接反应小肠细胞受自由基攻击损伤程度。光镜下观察肠道组织形态学变化证实肠道损伤程度评分进一步证实。Dreyer[4]和Curtis[5]报道心肌缺血再灌注过程中,中性粒细胞迁移并聚集在缺血再灌注的心肌,并且中性粒细胞活性和聚集程度的降低与心肌梗死面积的缩小有关。本实验表明缺血后处理小肠组织中MPO含量降低,表明缺血后处理减轻缺血组织的中性粒细胞的聚集,具有减轻小肠缺血再灌注损伤的作用。
总之,本实验表明肾脏缺血后处理能明显减轻小肠缺血再灌注损伤炎症反应。远隔缺血后处理可通过实施于非重要器官来保护某些对缺氧缺血极其敏感的重要器官,并且可在缺血事件发生后实施,因此,具有非常重要的临床应用价值。目前,RIP的研究尚处于实验探索阶段,动物实验研究发现,肾脏[1]、下肢[6]、颈总动脉[7]作为远隔器官,能减少心脏缺血面积,同样下肢短暂缺血也能减轻大脑[8]和对侧肢体[9]缺血再灌注损伤。Loukogeorgakis[10]等证明在人体中肢体短暂缺血具有减轻对侧肢体缺血再灌注后血管内皮细胞损伤。远隔器官缺血后处理的作用机制有待进一步研究。
参考文献
[1] Kerendi F, Kin H, HalkosME, et al.Remote postconditioning,brief renal ischemia and reperfusion applied before coronary artery reperfusion reduces myocardial infarct
size via endogenous activation of adenosine receptors.Basic Res Cardiol,2005,100(5):404-412.
[2] Chiu CJ, Mcardle AH, Brown R, et al. Intestinal mucosal lesion in low-flow states. Arch Surg,1970, 101:178-483.
[3] Li CM,Zhang XH,Ma XJ,et al.Limb ischemic postconditioning protects myocardium from ischemia-reperfusion injury.Scand Cardiovasc,2006,40(5):312-317.
[4] Dreyer WJ,Michael LH,West MS,et al.Neutrophil accumulation in ischemic canine myocardium.Insights into time course,distribution,and mechanism of localization during early reperfusion.Circulation,1991,84(1):400-411.
[5] Curtis WE,Gillinov AM,Wilson IC,et al.Inhibition of neutrophil adhesion reduces myocardial infarct size.Ann Thorac Surg,1993,56(5):1069-1072.
[6] Andreka G, Vertesaljai M, Szantho G,et al.Remote ischemic postconditioning protects the heart during acute myocardial infarction in pigs. Heart,2007,93(6):749-752.
[7] Gritsopoulos G,IIiodromitis EK,Zoga A,et al.Remote postconditioning is more potent than classic postconditioning in reducing the infarct size in anesthetized rabbits.Cardiovasc Drugs Ther,2009,23:193-198.
[8] Jaberansari MT,Baxter GF,Muller CA,et al.Limb remote ischemic postconditioning protects against focal ischemia in rats.Brain Res,2009,1288:88-94.
[9] Eberlin KR,McCormack MC,Nguyen JT,et al.Sequential limb ischemia demonstrates remote postconditioning protection of murine skeletal muscle.Plast Reconstr Surg,2009,123:8-16.
[10] Loukogeorgakis SP,Williams R,Panapiotidou AT,et al.Transient limb ischemia induces remote preconditioning and remote postconditioning in humans by a KATP-channel dependent mechanism.Circulation,2007,116(12):1386-1395.
【关键词】 远隔缺血后处理;缺血再灌注;急性肠缺血
Impact of remote postconditioning on rabbits with acute intestinal ischemia-reperfusion injury
CHUYong-xin, YANGMu, ZHANGju-wen.
Department of Vascular surgery,Yantai Yuhuangding Hospital,the Affiliated Hospital of medical College,Qingdao University,Shandong 264000,China
【Abstract】 Objective In this study, we tested the hypothesis that remote postconditioning induced by a single 5-min episode of renal artery occlusion and reperfusion applied immediately before the onset ofsuperior mesenteric artery reperfusion protects the intestinal from reperfusion injury. Methods 30 healthy New Zealand white rabbits were randomly divided into three groups(n=10 in each group): control, intestinal ischemic postconditioning (IIP), and renal ischemic postconditioning (RIP). Tissue myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were determined and the morphology of some intestinal tissues was observed by microscopy at the end of the experiment. Results MDA levels and MPO activity was significantly reduced in IIP and RIP as compared to control (P<0.01),also was confirmed by the score of intestinal mucosal injury. Conclusion Renal postconditioning provides potent intestinal mucosal injury. The potential mechanism of remote postconditioning might be associated decreasing the injury caused by oxygen free radicals and strengthening the action of antioxidation.
【Key words】 Remote postconditioning;Ischemia - reperfusion injury;Acute intestinal ischemia
DOI:10.3760/cma.j.issn 1673-8799.2010.11.46
作者单位:264000青岛大学医学院附属烟台毓璜顶医院血管外科
越来越多的肠系膜上动脉栓塞和血栓形成早期发现早期手术治疗肠缺血再灌注损伤越来越受到临床医生的重视。缺血预处理和缺血后适应均可减轻缺血再灌注的损伤。但无法预知组织及器官缺血具体时间,缺血预处理的临床应用受到了极大限制。缺血后适应的实施直接作用于已发生缺血的重要器官,在临床上不适用于对缺血极其敏感的器官。2005年Kerendi[1]等利用小鼠心肌缺血模型,在心脏缺血再灌注前对肾动脉进行单次5 min的闭塞和1 min再通,发现可显著减轻心肌缺血再灌注损伤,从而提出了远隔缺血后处理即:组织器官缺血再灌注前在非本组织器官短暂的缺血,能减轻缺血再灌注的损伤。本实验研究肠道缺血再灌注前肾动脉短暂缺血再灌注即肾脏缺血后处理对兔肠道缺血再灌注的影响,为临床寻找减轻肠道缺血再灌注损伤更佳方法提供实验依据。
1 材料和方法
1.1 实验动物
健康新西兰大白兔30只,雌雄不拘,体重1.5~2.0 kg。所有动物随机分为3组(每组10只):对照组(Con)、肠道缺血后处理组(IIP)、肾脏缺血后处理组(RIP)。
1.2 方法
1.2.1 手术过程 所有新西兰兔术前12 h禁食,自由进水。称重后左下腹腔内注射10%水合氯醛(3 ml/kg)麻醉。上腹正中切口,长约10 cm,进腹后予肠系膜根部仔细分离显露肠系膜上动脉,再仔细分离显露左肾动脉,用无创小血管夹阻断动脉,分组干预:对照组:阻断肠系膜上动脉30 min,再灌注120 min; 肠道缺血后处理组:阻断肠系膜上动脉30 min 后,灌注1 min,阻断1 min,反复3次,继而在灌注到120 min; 肾脏缺血后处理组: 阻断肠系膜上动脉24 min 时,阻断左肾动脉5 min,开放肾动脉1 min,再灌注肠系膜上动脉120 min。
1.2.2 标本采集 再灌注120 min后直视下下腔静脉穿刺取血5 ml,血标本收集后,常温下静置30 min, 3000r/min离心15 min,分离的血浆保存于-20℃冰箱,待所有标本收集完后一次性检测。分光度计测定血浆MDA水平,血浆MDA含量以每毫升血浆所含的数量来表示(mmol/g)。在无菌原则下取距回盲部约20 cm的近端小肠组织5 cm左右,切除的近端2.5 cm肠组织冷冻贮存于-80℃,分光度计测定小肠组织MPO活性,并以每百克组织所含的单位来表示(U/100 g)。试剂盒均购于南京建成生物工程研究所。远端2.5 cm肠组织用40 g/L甲醛溶液固定,用石蜡包埋,切片,苏木精-伊红染色行组织病理学检查。肠组织病理评分采用双盲法按Chiu6级[2]评分法分析。
1.2.3 统计学方法 采用SPSS 11.0软件进行统计学处理,计量资料采用t检验,组间比较采用单因素方差分析,P<0.05为差异有统计学意义。
2 结果
2.1 将各组血浆中MDA的含量和组织中MPO的活性测定严格按照试剂盒说明进行操作,双盲法测得的肠壁损伤病理评分,见表1。SIP组和RIP组的MDA含量、MPO的活性及肠黏膜损伤评分与Con组比较,差异均有显著性(P<0.01)。SIP组与RIP组的MDA含量、MPO的活性及肠黏膜损伤评分比较无显著差异(P>0.0 5)。
3 讨论
本实验发现兔小肠急性缺血再灌注前给予肾动脉短暂缺血再灌可明显减轻小肠的缺血再灌注损伤。Li[3]等在远隔缺血后处理对心肌缺血的保护实验中发现丙二醛活性显著降低,提示远隔缺血后处理可减轻缺血心肌的过氧化反应。本实验血浆MDA含量较对照组明显减少,提示肾脏缺血后处理可减轻小肠缺血再灌注损伤的脂质过氧化,间接反应小肠细胞受自由基攻击损伤程度。光镜下观察肠道组织形态学变化证实肠道损伤程度评分进一步证实。Dreyer[4]和Curtis[5]报道心肌缺血再灌注过程中,中性粒细胞迁移并聚集在缺血再灌注的心肌,并且中性粒细胞活性和聚集程度的降低与心肌梗死面积的缩小有关。本实验表明缺血后处理小肠组织中MPO含量降低,表明缺血后处理减轻缺血组织的中性粒细胞的聚集,具有减轻小肠缺血再灌注损伤的作用。
总之,本实验表明肾脏缺血后处理能明显减轻小肠缺血再灌注损伤炎症反应。远隔缺血后处理可通过实施于非重要器官来保护某些对缺氧缺血极其敏感的重要器官,并且可在缺血事件发生后实施,因此,具有非常重要的临床应用价值。目前,RIP的研究尚处于实验探索阶段,动物实验研究发现,肾脏[1]、下肢[6]、颈总动脉[7]作为远隔器官,能减少心脏缺血面积,同样下肢短暂缺血也能减轻大脑[8]和对侧肢体[9]缺血再灌注损伤。Loukogeorgakis[10]等证明在人体中肢体短暂缺血具有减轻对侧肢体缺血再灌注后血管内皮细胞损伤。远隔器官缺血后处理的作用机制有待进一步研究。
参考文献
[1] Kerendi F, Kin H, HalkosME, et al.Remote postconditioning,brief renal ischemia and reperfusion applied before coronary artery reperfusion reduces myocardial infarct
size via endogenous activation of adenosine receptors.Basic Res Cardiol,2005,100(5):404-412.
[2] Chiu CJ, Mcardle AH, Brown R, et al. Intestinal mucosal lesion in low-flow states. Arch Surg,1970, 101:178-483.
[3] Li CM,Zhang XH,Ma XJ,et al.Limb ischemic postconditioning protects myocardium from ischemia-reperfusion injury.Scand Cardiovasc,2006,40(5):312-317.
[4] Dreyer WJ,Michael LH,West MS,et al.Neutrophil accumulation in ischemic canine myocardium.Insights into time course,distribution,and mechanism of localization during early reperfusion.Circulation,1991,84(1):400-411.
[5] Curtis WE,Gillinov AM,Wilson IC,et al.Inhibition of neutrophil adhesion reduces myocardial infarct size.Ann Thorac Surg,1993,56(5):1069-1072.
[6] Andreka G, Vertesaljai M, Szantho G,et al.Remote ischemic postconditioning protects the heart during acute myocardial infarction in pigs. Heart,2007,93(6):749-752.
[7] Gritsopoulos G,IIiodromitis EK,Zoga A,et al.Remote postconditioning is more potent than classic postconditioning in reducing the infarct size in anesthetized rabbits.Cardiovasc Drugs Ther,2009,23:193-198.
[8] Jaberansari MT,Baxter GF,Muller CA,et al.Limb remote ischemic postconditioning protects against focal ischemia in rats.Brain Res,2009,1288:88-94.
[9] Eberlin KR,McCormack MC,Nguyen JT,et al.Sequential limb ischemia demonstrates remote postconditioning protection of murine skeletal muscle.Plast Reconstr Surg,2009,123:8-16.
[10] Loukogeorgakis SP,Williams R,Panapiotidou AT,et al.Transient limb ischemia induces remote preconditioning and remote postconditioning in humans by a KATP-channel dependent mechanism.Circulation,2007,116(12):1386-1395.